A Transcription Factor Functional Atlas of Germline Development

A Transcription Factor Functional Atlas of Germline Development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Resource A Transcription Factor Functional Atlas of Germline Development Roger Pocock, Wei Cao, Qi Fan, Gemmarie Amparado, Dean Begic, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3880498/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Aug, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Fertility requires the faithful proliferation of germ cells and their differentiation into gametes. Controlling these cellular states demands precise timing and expression of gene networks. Transcription factors (TFs) play critical roles in gene expression networks that influence germ cell development. There has, however, been no functional analysis of the entire TF repertoire in controlling in vivo germ cell development. Here, we analyzed germ cell states and germline architecture to systematically investigate the function of 364 germline-expressed TFs in the Caenorhabditis elegans germ line. Using germline-specific knockdown, automated germ cell counting, and high-content analysis of germ cell nuclei and plasma membrane organization, we identify 156 TFs with discrete autonomous germline functions. By identifying TFs that control the germ cell cycle, proliferation, differentiation, germline structure and fertility, we have created an atlas for mechanistic dissection of germ cell behavior and gamete production. Biological sciences/Developmental biology/Germline development Biological sciences/Molecular biology/Transcription Biological sciences/Developmental biology/Experimental organisms/Model invertebrates/Caenorhabditis elegans Germ line gametes transcription factors RNA interference Caenorhabditis elegans Figures Figure 1 Figure 2 Figure 3 Figure 4 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files FigureS1.pdf FigureS2.pdf FigureS3.pdf FigureS4.pdf FigureS5.pdf FigureS6.pdf FigureS7.pdf FigureS8.pdf TableS1.Germlinetranscriptomeanalysis.xlsx Table S1 TableS2WormGTFexpressionandreproductivefunctions.xlsx Table S2 TableS3RNAiplasmidsusedinthisstudy.xlsx Table S3 TableS4Distalgermlineanalysis.xlsx Table S4 TableS5ProximalgermlineanalysisV6.xlsx Table S5 TableS6156GTFswithgermlinefunctions.xlsx Table S6 TableS7PreviouslyreportedphenotypesinC.elegans.xlsx Table S7 TableS8GTFfunctionsinotherorganismsGOanalysis.xlsx Table S8 TableS9Strainsusedinthisstudy.xlsx Table S9 TableS10Oligosusedinthisstudy.xlsx Table S10 TableS11Sourcedata.xlsx Table S11 Cite Share Download PDF Status: Published Journal Publication published 11 Aug, 2024 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3880498","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Resource","associatedPublications":[],"authors":[{"id":269293153,"identity":"c285782f-da98-420c-a179-ad64d7a7bbd6","order_by":0,"name":"Roger Pocock","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYJADZoYPYJoHiNmI1MI4g2QtzDzEaDFvb7/4uILhjhx/e/NjY9scmzz+/rMHGD6UHcapRebMmWLDMwzPjCXOHDNOzt2WVixxIy+BccY53FokJHLSJBsYDidukMhhPpy77XBiww0eA2beNrxa0n/CtVgCtcw/f8aA+S9eLenHGGFakhmBWjYcyDFgZsSnhecMs2SDAcQvhr3b0hI33sgxONhzLh23Fvb2hx8bKiAhJvFzm03ivPNnDB/8KLPGqQUYCwYMDAYHUMUOYFOIAOwPCKsZBaNgFIyCkQ0AeAtXe4d+AWIAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-5515-3608","institution":"Monash University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Roger","middleName":"","lastName":"Pocock","suffix":""},{"id":269293154,"identity":"fb510137-e882-42ec-a6b2-e0a84168ea8a","order_by":1,"name":"Wei Cao","email":"","orcid":"","institution":"Monash University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Cao","suffix":""},{"id":269293155,"identity":"2c6c6ef8-8fa8-4ebc-8d79-bc352d54a708","order_by":2,"name":"Qi Fan","email":"","orcid":"","institution":"Monash University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Fan","suffix":""},{"id":269293156,"identity":"32b5562f-db64-4baf-980e-0d59755089cb","order_by":3,"name":"Gemmarie Amparado","email":"","orcid":"","institution":"Monash University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gemmarie","middleName":"","lastName":"Amparado","suffix":""},{"id":269293157,"identity":"e02b0c57-f4de-4996-a7a6-3b6541fa0182","order_by":4,"name":"Dean Begic","email":"","orcid":"","institution":"Monash University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dean","middleName":"","lastName":"Begic","suffix":""},{"id":269293158,"identity":"9113e1f5-0f4e-4f4e-a730-3507f236e60a","order_by":5,"name":"Rasoul Godini","email":"","orcid":"","institution":"Monash University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rasoul","middleName":"","lastName":"Godini","suffix":""},{"id":269293159,"identity":"288279e6-7d1c-41aa-a667-8d173c26016a","order_by":6,"name":"Sandeep Gopal","email":"","orcid":"https://orcid.org/0000-0002-6706-6747","institution":"[email protected]","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Sandeep","middleName":"","lastName":"Gopal","suffix":""}],"badges":[],"createdAt":"2024-01-20 03:20:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3880498/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3880498/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-024-51212-0","type":"published","date":"2024-08-11T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50231304,"identity":"e767098f-d5ce-4cbb-bc53-b47879079f2d","added_by":"auto","created_at":"2024-01-26 21:33:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6107901,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic Profiling of Germline Transcription Factor Function\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(A) Schematic of the C. elegans hermaphrodite germline (left), identification of germline-expressed TFs by RNA sequencing (center), and representative images and phenotypic readouts for high-content distal and proximal germline analysis (right). The distal germ line is labelled with DAPI (nuclei), SYGL-1 (Notch target gene) and pH3 (M-phase chromosomes). PZ = progenitor zone, TZ = transition zone. The proximal germ line is marked with transgenic fluorophores: red = nuclei; green = plasma membrane. Scale bars = 20 µm.\u003c/p\u003e\n\u003cp\u003e(B-D) Heatmaps showing phenotypic overview for TF silencing causing distal (B ≥20% change compared to control), proximal (C ≥50% of animals with the phenotype) or distal and proximal germline phenotypes (D). (B) TF family categories (colored boxes); progenitor zone (PZ), transition zone (TZ) and SYGL-1+ region phenotypes shown as percentage change compared to control (blue = decrease; red = increase); germline expression of each TF (grey bars = log2 CPM); mammalian ortholog associated with the reproduction GO term (blue boxes = No, black boxes = Yes). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(C) Labelling as in B, except purple = proximal phenotype; blue = no phenotype detected. Phenotypes shown in this figure: small germ line, rachis defect (narrow or wide rachis), meiotic defect (multinucleated cells, abnormal meiotic progression), apoptosis, oocyte defect (delayed expansion at the turn, difference in single-array oocyte number, difference in budded oocyte number, vesiculation), sperm defect (mislocated sperm, incomplete spermatogenesis). (D) Labelling as in B and C. Note: the heatmaps do not include essential GTFs identified in the screens – this analysis is detailed in Figure 4 and Table S4.\u003c/p\u003e\n\u003cp\u003e(E) TF family distribution of the 875 C. elegans TFs, 96 TFs that regulate the distal germ line or are essential for germline development, and 52 TFs that regulate the proximal germline.\u003c/p\u003e\n\u003cp\u003eTF families shown in this figure: bZIP = basic leucine zipper domain, HD = homeodomain, HMG = High mobility group box domain, MYB= myeloblastosis viral oncogene homolog, NHR = nuclear hormone receptors, T-box, WH = winged helix, ZF = zinc finger.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3880498/v1/06697e88390f4d4394c48ef3.png"},{"id":50231303,"identity":"001d0b40-d973-4453-9520-adcfbd8a4fa6","added_by":"auto","created_at":"2024-01-26 21:33:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":326273,"visible":true,"origin":"","legend":"\u003cp\u003eTF Control of Distal Germ Cell Behavior\u003c/p\u003e\n\u003cp\u003e(A) Quantification of nuclei number in the PZ of 3xflag::sygl-1; sun-1p::rde-1; rde-1(mkc36) one-day adults. n = 23-24. Green dashed line = average of the control.\u003c/p\u003e\n\u003cp\u003e(B and C) Quantification of nuclei number (B) and confocal micrographs (C) of one-day adult germline PZ of sun-1p::rde-1; rde-1(mkc36) treated with control RNAi and glp-1(ar202); sun-1p::rde-1; rde-1(mkc36) treated with control or experimental RNAi. n = 29-34.\u003c/p\u003e\n\u003cp\u003e(D and E) Quantification of SYGL-1+ nuclei number (D) and confocal micrographs of germline PZ\u003c/p\u003e\n\u003cp\u003e(E) of 3xflag::sygl-1; sun-1p::rde-1; rde-1(mkc36) one-day adults. n = 23-24. Green dashed line = average of the control (D); yellow dashed line = PZ/TZ boundary (E - left) and white line = SYGL-1 boundary (E - right).\u003c/p\u003e\n\u003cp\u003e(F) Proliferation rates of the C. elegans germ line after GTF RNAi. RNAi was performed from the L1 stage for 66 hrs before commencing EdU labelling, and germ lines collected and imaged after 4 and 10 hrs. The 3xflag::sygl-1; sun-1p::rde-1; rde-1(mkc36) strain was used in this experiment. Results of three independent experiments were shown. n = 9-13 for each experiment.\u003c/p\u003e\n\u003cp\u003e(G) Confocal micrographs showing DAPI and EdU+ nuclei after 4 and 10 hrs of EdU labelling.\u003c/p\u003e\n\u003cp\u003e(H) Heatmap showing the effect of silencing 11 GTF on proliferation rate, mitotic index, and nuclei numbers of PZ, TZ, SYGL-1+ and pH3+.\u003c/p\u003e\n\u003cp\u003eRNAi was performed from the L1 stage. Data was generated from three independent experiments, and results were normalized to respective controls in (A) (B) and (D). P values assessed comparing to control RNAi by multiple unpaired t-test with no correction for multiple comparison (A, B, D and F). Error bars indicate SEM. Scale bars = 20 µm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3880498/v1/73923ea5224526215269f316.png"},{"id":50231301,"identity":"69dcb5d1-c864-4d57-b55c-0d7e8763408e","added_by":"auto","created_at":"2024-01-26 21:33:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1032966,"visible":true,"origin":"","legend":"\u003cp\u003eTF Control of Meiotic Germ Cell Behavior\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(A)\tQuantification of multinucleated germ cells of pie-1p::mCherry::his-58; pie-1p::GFP::PH(PLC1delta1); sun-1p::rde-1; rde-1(mkc36) two-day adults following RNAi from the L1 stage. Data was generated from three independent experiments. n = 30-31. P values assessed by one-way ANOVA with no correction for multiple comparison.\u003c/p\u003e\n\u003cp\u003e(B)\tFluorescence micrographs of wild-type or ced-3(rp190) two-day adult germ lines in the pie-1p::mCherry::his-58; pie-1p::GFP::PH(PLC1delta1); sun-1p::rde-1; rde-1(mkc36) strain. Control RNAi and RNAi of dmd-7 and baz-2 were applied from the L1 stage in wild-type animals. Dash lines = border between the TZ (above) and pachytene region (below) of the germ line. Asterisks = multinucleated cells; triangles = apoptotic cells. Scale bar = 20 µm.\u003c/p\u003e\n\u003cp\u003e(C and D) Quantification of apoptotic germ cells (C) and multinucleated germ cells (D) in two-day adults of pie-1p::mCherry::his-58; pie-1p::GFP::PH(PLC1delta1); sun-1p::rde-1; rde-1(mkc36) strain (wild-type and ced-3(rp190) animals). Data generated from three independent experiments. n = 30-33. P values assessed by unpaired t-test.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(E) Quantification of multinucleated germ cells in ced-3(rp190), pie-1p::mCherry::his-58; pie-1p::GFP::PH(PLC1delta1); sun-1p::rde-1; rde-1(mkc36) two-day adults following RNAi from the L1 stage. Data generated from three independent experiments. n = 30. P values assessed by one-way ANOVA with no correction for multiple comparison.\u003c/p\u003e\n\u003cp\u003e(F and G) Confocal micrographs of DAPI (white) and phalloidin (pink) staining (F) and quantification of germline folding events in the pachytene region (G) of sun-1p::rde-1; rde-1(mkc36) one-day adults. Images for control RNAi (L4440) and lin-26 RNAi are shown in F (note - only one plane is shown). Data was generated from four independent experiments. n = 24-36. Yellow dashed line = border between the TZ (right) and pachytene region (left) of the germ line. P values assessed by one-way ANOVA with no correction for multiple comparison.\u003c/p\u003e\n\u003cp\u003eRNAi was performed from the L1 stage. Error bars indicate SEM. Scale bars = 20 µm.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3880498/v1/88e8f4d564231cb87e56abf9.png"},{"id":50231639,"identity":"da163edd-bdca-4b37-93dc-e2ab7d14b566","added_by":"auto","created_at":"2024-01-26 21:41:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":318879,"visible":true,"origin":"","legend":"\u003cp\u003eEssential TFs act Late in Germline Development to Control Fertility\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(A)\tQuantification of hatched larvae and dead eggs (brood size) following germline-specific RNAi from the L1 stage. Data generated from three independent experiments. n = 15-18.\u003c/p\u003e\n\u003cp\u003e(B)\tTimeline of temporal germline analysis showing the time and respective life stages when germline imaging and analysis were performed following egg-laying on control and RNAi plates (RNAi from the L1 stage).\u003c/p\u003e\n\u003cp\u003e(C)\tQuantification of germline length of pie-1p::mCherry::his-58; sun-1p::rde-1; rde-1(mkc36) animals at the L4 stage following RNAi from the L1 stage. Data was generated from three independent experiments, and results normalized to respective controls. n = 26-30. P values assessed by one-way ANOVA with no correction for multiple comparison. Error bars indicate SEM.\u003c/p\u003e\n\u003cp\u003e(D)\tDIC (left) and fluorescent micrographs (right) of pie-1p::mCherry::his-58; sun-1p::rde-1; rde-1(mkc36) germ lines at the L4 stage following control, cdc-5L and znf-622 RNAi. Blue arrow = vulva. Scale bar = 20 µm.\u003c/p\u003e\n\u003cp\u003e(E)\tHeatmap showing the percentage of germ lines producing sperm, oocytes and embryos in day 1, day 2 and day 3 of adulthood following germline-specific RNAi from the L1 stage. For day 2 and day 3 adult analysis, worms were selected from day 1 sterile animals, except for the control group, and incubated for another 48 hrs or 72hrs prior to analysis. Data was generated from three independent experiments. n = 30.\u003c/p\u003e\n\u003cp\u003e(F) Quantification of hatched larvae and dead eggs (brood size) following germline-specific RNAi from the L4 stage. Data generated from three independent experiments. n = 17-18. P values assessed by one-way ANOVA with no correction for multiple comparison.\u003c/p\u003e\n\u003cp\u003e(G-J) Quantification of nuclei number of PZ (G), TZ (H), pH3+ (I) and SYGL-1+ (J) of 3xflag::sygl-1; sun-1p::rde-1; rde-1(mkc36) one-day adult germ lines following RNAi treatment from the L4 stage. n = 22-24.\u003c/p\u003e\n\u003cp\u003eP values assessed by one-way ANOVA with no correction for multiple comparison. Error bars indicate SEM.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3880498/v1/3fcdbc2869540c7cccd0fe2c.png"},{"id":62254971,"identity":"0dd9dee1-d7f0-4f23-bd73-2c5f38b49660","added_by":"auto","created_at":"2024-08-12 07:06:54","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15207770,"visible":true,"origin":"","legend":"","description":"","filename":"Caoetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3880498/v1_covered_622100cf-0264-4466-b6f8-11a49910970f.pdf"},{"id":50231305,"identity":"0a804483-353c-4831-8565-42a10c079ded","added_by":"auto","created_at":"2024-01-26 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Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Germ line, gametes, transcription factors, RNA interference, Caenorhabditis elegans","lastPublishedDoi":"10.21203/rs.3.rs-3880498/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3880498/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Fertility requires the faithful proliferation of germ cells and their differentiation into gametes. Controlling these cellular states demands precise timing and expression of gene networks. \r\nTranscription factors (TFs) play critical roles in gene expression networks that influence germ cell development. There has, however, been no functional analysis of the entire TF repertoire in controlling in vivo germ cell development. Here, we analyzed germ cell states and germline architecture to systematically investigate the function of 364 germline-expressed TFs in the \u003ci\u003eCaenorhabditis elegans\u003c/i\u003e germ line. Using germline-specific knockdown, automated germ cell counting, and high-content analysis of germ cell nuclei and plasma membrane organization, we identify 156 TFs with discrete autonomous germline functions. By identifying TFs that control the germ cell cycle, proliferation, differentiation, germline structure and fertility, we have created an atlas for mechanistic dissection of germ cell behavior and gamete production.","manuscriptTitle":"A Transcription Factor Functional Atlas of Germline Development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-26 21:33:48","doi":"10.21203/rs.3.rs-3880498/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"47bf47bc-dbdf-4a8e-a1da-647815c88459","owner":[],"postedDate":"January 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":28377095,"name":"Biological sciences/Developmental biology/Germline development"},{"id":28377096,"name":"Biological sciences/Molecular biology/Transcription"},{"id":28377097,"name":"Biological sciences/Developmental biology/Experimental organisms/Model invertebrates/Caenorhabditis elegans"}],"tags":[],"updatedAt":"2024-08-12T07:06:07+00:00","versionOfRecord":{"articleIdentity":"rs-3880498","link":"https://doi.org/10.1038/s41467-024-51212-0","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2024-08-11 04:00:00","publishedOnDateReadable":"August 11th, 2024"},"versionCreatedAt":"2024-01-26 21:33:48","video":"","vorDoi":"10.1038/s41467-024-51212-0","vorDoiUrl":"https://doi.org/10.1038/s41467-024-51212-0","workflowStages":[]},"version":"v1","identity":"rs-3880498","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3880498","identity":"rs-3880498","version":["v1"]},"buildId":"CiT4i_kKBbxQbnFL0ufpk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}